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Biochemical Assays for MTase Activity
生物化学分析法检测甲基转移酶活性   

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Abstract

Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is essential for cell and virus. Guanine-N7-methyltransferase (N7-MTase) methylates the GpppN cap at the N7 position of guanine, resulting in cap-0 structure (m7GpppN), and Ribose 2'-O-MTase further methylates the first nucleotide of higher eukaryotic cellular and viral mRNAs at the ribose 2'-OH position to form cap-1 (m7GpppNm) structures. Here, we describe a biochemical assay to detect the activities of mRNA capping MTases.

Keywords: Methyltransferase(甲基转移酶), S-adenosyl-L-methionine(S-腺苷甲硫氨酸), RNA capping(RNA封盖), Cap structure(盖结构)

Materials and Reagents

  1. Bodicon m7G capping system (Bodicon, catalog number: CS0130 )
  2. S-adenosyl methionine (SAM) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
  3. Bodicon Capping Enzyme (10 U/µl) (involved in Bodicon m7G capping system) (Bodicon, catalog number: CS0130)
    Note: Because the sale of this kit was low, the previous companies which provided this capping kit were out of service. This capping kit was provided by a new company in China as custom-made products (contact e-mail: service@bodicon.cn, phone:+86-13628662011). In fact the similar capping kit from any other companies (such as EPICENTRE biotechnologies, ScriptCap m7G capping system, catalog number: SCCE0610 ) is suitable for this experiment, and people can also contact with us to get the related protein or kit.
  4. Inorganic pyrophospatase (YIPP) (New England Biolabs, catalog number: M2403S )
  5. S-adenosyl [methyl-3H] methionine ([3H]-SAM) (PerkinElmer, catalog number: NET155H001MC )
  6. DEAE Sephadex (GE Healthcare, catalog number: 17-0170-01 )
  7. GTP (Thomas Scientific, catalog number: R0461 )
  8. RNase inhibitor (Thomas Scientific, catalog number: EO0381 )
  9. RNase free water
  10. Phenol-chloroform (pH 4.8-5.2 for RNA only)
  11. Ethanol (RNase free)
  12. RNase free water
  13. Sodium Dodecyl Sulfonate (SDS)
  14. Ethylene Diamine Tetraacetic Acid (EDTA)
  15. NH4HCO3
  16. NaCl
  17. 10x MTase Buffer (see Recipes)
  18. Cap-0 cap structure (m7GpppN-RNA) (see Recipes)
  19. Non-methylated Cap-0 cap structure (GpppN-RNA) (see Recipes)
  20. MTase assay reaction mix (see Recipes)

Equipment

  1. Bechtop
  2. Water bath
  3. Centrifuge
  4. Liquid scintillation counter

Procedure

  1. Synthesis of RNA cap structure as substrates.
    The nascent RNA transcribed in vitro possesses a 5' triphosphate end. Two RNA capping systems are used to synthesize cap structure as the substrates of MTase.
    1. Combine 1-10 µg RNA and RNase free water up to 12.5 µl of total reaction volume.
    2. To heat to denature the in vitro transcribed RNA, incubate the tube at 65 °C for 10 min, and transfer the tube to ice immediately.
    3. Combine the following reaction components in the order given:
      Cap-0 cap structure (m7GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      3 mM SAM   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
      Non-methylated Cap-0 cap structure (GpppN-RNA)
      Heat denatured RNA   
      12.5 µl
      10x Bodicon Capping Buffer   
      2 µl
      10 mM GTP   
      2 µl
      Inorganic pyrophospatase 0.1 U/µl   
      1 µl
      RNase inhibitor 40 U/µl   
      0.5 µl
      Bodicon Capping Enzyme (10 U/µl)   
      2 µl
      Total   
      20 µl
    4. Incubate at 37 °C for 2 h.
    5. Purify the RNA substrates by using phenol-chloroform extraction and ethanol precipitation methods.
  2. Prepare 10x MTase Buffer. The MTase buffer may be changed depending on different interested MTases.
  3. Combine the following reaction components in the order given for MTase assays:
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water
    up to 30 μl
    Total
    30 μl
  4. Incubate the reaction at 30-37 °C depending on different MTases for 1.5 h.
  5. Transfer the tubes onto ice and add equal volume (30 μl) of 0.2% SDS, 20 mM EDTA.
  6. Keep the tubes on ice, add 1 ml of 10 mM NH4HCO3 (pH 8.5).
  7. Prepare 1 ml DEAE Sephadex column and equilibrated with 10 ml 10 mM NH4HCO3 (pH 8.5).
  8. Load the samples onto the equilibrated column.
  9. Wash the column with 10 ml of 10 mM NH4HCO3 (pH 8.5), 100 mM NaCl.
  10. Elute the samples with 1.5 ml of 10 mM NH4HCO3 (pH 8.5), 400 mM NaCl.
  11. Add equal volume scintillation liquid, mix well by vortexing and measure the signal using Liquid scintillation counter. The counting signal of [3H], which is transformed from [3H]-SAM to RNA substrates, represents the activity of tested MTases.

Recipes

  1. 10x MTase Buffer
    0.5 M Tris-HCl (pH 7.5 or 8.0)
    50 mM KCl
    20 mM MgCl2
    20 mM DTT
  2. Cap-0 cap structure (m7GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    3 mM SAM
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  3. Non-methylated Cap-0 cap structure (GpppN-RNA)
    Heat denatured RNA
    13.5 µl
    10x Bodicon Capping Buffer
    2 µl
    10 mM GTP
    2 µl
    Inorganic pyrophospatase 0.1 U/µl
    1 µl
    RNase inhibitor 40 U/µl
    0.5 µl
    Bodicon Capping Enzyme (10 U/µl)
    2 µl
    Total
    20 µl
  4. MTase assay reaction mix
    Purified MTases
    1 µg
    10x MTase Buffer
    3 μl
    GpppN-RNA or m7GpppN-RNA
    2 μg
    3 mM SAM
    0.5 μl
    [3H]-SAM (67.3 Ci/mmol, 0.5 μCi/μl)
    1 μl
    RNase inhibitor 40 U/μl
    1 μl
    RNase free water up to
    30 μl
    Total
    30 μl

Acknowledgments

We thank Dr. Tero Ahola from University of Helsinki for the kindly help and advices for setting up this MTase assay. We thankfully acknowledge the University of Helsinki for providing research facilities and support during our visits. This work was supported by the China NSFC grants (81130083, 31170152 and 81271817).

References

  1. Chen, Y., Tao, J., Sun, Y., Wu, A., Su, C., Gao, G., Cai, H., Qiu, S., Wu, Y., Ahola, T. and Guo, D. (2013). Structure-function analysis of severe acute respiratory syndrome coronavirus RNA cap guanine-N7-methyltransferase. J Virol 87(11): 6296-6305.
  2. Chen, Y., Su, C., Ke, M., Jin, X., Xu, L., Zhang, Z., Wu, A., Sun, Y., Yang, Z., Tien, P., Ahola, T., Liang, Y., Liu, X. and Guo, D. (2011). Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog 7(10): e1002294.
  3. Chen, Y., Cai, H., Pan, J., Xiang, N., Tien, P., Ahola, T. and Guo, D. (2009). Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc Natl Acad Sci U S A 106(9): 3484-3489.

简介

甲基转移酶(MTase)将甲基(-CH 3)从供体S-腺苷-L-甲硫氨酸(AdoMet或SAM)转移到生物活性分子例如激素,神经递质,脂质,蛋白质和核酸。 加入甲基引起分子的物理化学性质的改变。 mRNA帽结构对于细胞和病毒是必需的。 鸟嘌呤-N7-甲基转移酶(N7-MTase)甲基化在鸟嘌呤的N7位置的GpppN帽,导致cap-0结构(m7GpppN),核糖2'-O-MTase进一步甲基化高等真核细胞和病毒的第一个核苷酸 mRNA在核糖2'-OH位置形成cap-1(m7GpppNm)结构。 在这里,我们描述一种生物化学测定检测mRNA上限MTases的活动。

关键字:甲基转移酶, S-腺苷甲硫氨酸, RNA封盖, 盖结构

材料和试剂

  1. Bodicon m7G加帽系统(Bodicon,目录号:CS0130)
  2. S-腺苷甲硫氨酸(SAM)(参与Bodicon m7G加帽系统)(Bodicon,目录号:CS0130)
  3. Bodicon加帽酶(10U /μl)(参与Bodicon m7G加帽系统)(Bodicon,目录号:CS0130)
    注意:由于这个套件的销售很低,提供这个套件的以前的公司停止服务。这种封盖套件由中国的一家新公司作为定制产品提供(联系电子邮件:service@bodicon.cn,电话:+ 86-13628662011)。事实上,来自任何其他公司(例如EPICENTRE生物技术,ScriptCap m7G加帽系统,目录号:SCCE0610)的类似的封端试剂盒适合于该实验,人们也可以与我们联系以获得相关的蛋白质或试剂盒。
  4. 无机焦磷酸化酶(YIPP)(New England Biolabs,目录号:M2403S)
  5. S-腺苷[甲基 - [3 H]甲硫氨酸([3 H] -SAM)(PerkinElmer,目录号:NET155H001MC)
  6. DEAE Sephadex(GE Healthcare,目录号:17-0170-01)
  7. GTP(Thomas Scientific,目录号:R0461)
  8. RNase抑制剂(Thomas Scientific,目录号:EO0381)
  9. 无RNase水
  10. 苯酚 - 氯仿(仅对于RNA,pH 4.8-5.2)
  11. 乙醇(不含RNase)
  12. 无RNase水
  13. 十二烷基磺酸钠(SDS)
  14. 乙二胺四乙酸(EDTA)
  15. NH 4 HCO 3 sub。
  16. NaCl
  17. 10x MTase Buffer(参见配方)
  18. Cap-0帽结构(m7GpppN-RNA)(参见配方)
  19. 非甲基化Cap-0帽结构(GpppN-RNA)(参见配方)
  20. MTase测定反应混合物(参见配方)

设备

  1. Bechtop
  2. 水浴
  3. 离心机
  4. 液体闪烁计数器

程序

  1. 合成RNA帽结构作为底物 在体外转录的新生RNA具有5'三磷酸末端。 两个RNA加帽系统用于合成帽结构作为MTase的底物。
    1. 结合1-10微克RNA和无RNase水,总反应体积为12.5微升
    2. 为了加热变性体外转录的RNA,将管在65℃下孵育10分钟,并立即将管转移到冰中。
    3. 按给定的顺序组合以下反应组分:
      Cap-0帽结构(m7GpppN-RNA)
      热变性RNA   
      12.5μl
      10x Bodicon Capping Buffer   
      2微升
      10 mM GTP   
      2微升
      3 mM SAM   
      1微升
      RNase inhibitor 40 U /μl   
      0.5μl
      Bodicon封顶酶(10 U /μl)   
      2微升
      总计   
      20微升
      非甲基化Cap-0帽结构(GpppN-RNA)
      热变性RNA   
      12.5μl
      10x Bodicon Capping Buffer   
      2微升
      10 mM GTP   
      2微升
      无机焦磷酸酶0.1 U /μl   
      1微升
      RNase inhibitor 40 U /μl   
      0.5μl
      Bodicon封顶酶(10 U /μl)   
      2微升
      总计   
      20微升
    4. 在37℃孵育2小时
    5. 通过使用苯酚 - 氯仿萃取和乙醇沉淀方法纯化RNA底物
  2. 准备10x MTase缓冲液。 MTase缓冲区可以根据不同的感兴趣的MTases而改变
  3. 按照为MTase测定给出的顺序组合以下反应组分:
    纯化的MTases
    1微克
    10x MTase Buffer
    3微升
    GpppN-RNA或m7GpppN-RNA
    2微克
    3 mM SAM
    0.5μl
    [ε3 H] -SAM(67.3Ci/mmol,0.5μCi/μl)
    1微升
    核糖核酸酶抑制剂40 U /μl
    1微升
    无RNase水
    最多30μl
    总计
    30微升
  4. 根据不同的MTase,在30-37℃孵育反应1.5小时
  5. 将管转移到冰上,加入等体积(30μl)的0.2%SDS,20mM EDTA
  6. 将管保持在冰上,加入1ml 10mM NH 4 HCO 3(pH8.5)。
  7. 制备1ml DEAE Sephadex柱,并用10ml 10mM NH 4 HCO 3(pH8.5)平衡。
  8. 将样品装入平衡柱。
  9. 用10ml 10mM NH 4 HCO 3(pH8.5),100mM NaCl洗涤柱子。
  10. 用1.5ml 10mM NH 4 HCO 3(pH8.5),400mM NaCl洗脱样品。
  11. 加入等体积闪烁液,涡旋混匀,用液体闪烁计数器测量信号。 从[3 H] -SAM转化为RNA底物的[3 H] H的计数信号代表测试的MT酶的活性。

食谱

  1. 10x MTase Buffer
    0.5 M Tris-HCl(pH 7.5或8.0)
    50 mM KCl
    20mM MgCl 2/
    20 mM DTT
  2. Cap-0帽结构(m7GpppN-RNA)
    热变性RNA
    13.5微升
    10x Bodicon加盖缓冲区
    2微升
    10 mM GTP
    2微升
    3 mM SAM
    1微升
    核糖核酸酶抑制剂40 U /μl
    0.5μl
    Bodicon封顶酶(10 U /μl)
    2微升
    总计
    20微升
  3. 非甲基化Cap-0帽结构(GpppN-RNA)
    热变性RNA
    13.5微升
    10x Bodicon加盖缓冲区
    2微升
    10 mM GTP
    2微升
    无机焦磷酸酶0.1 U /μl
    1微升
    核糖核酸酶抑制剂40 U /μl
    0.5μl
    Bodicon封顶酶(10 U /μl)
    2微升
    总计
    20微升
  4. MTase测定反应混合物
    纯化的MTases
    1微克
    10x MTase Buffer
    3微升
    GpppN-RNA或m7GpppN-RNA
    2微克
    3 mM SAM
    0.5μl
    [ε3 H] -SAM(67.3Ci/mmol,0.5μCi/μl)
    1微升
    核糖核酸酶抑制剂40 U /μl
    1微升
    无RNase的水达到
    30微升
    总计
    30微升

致谢

我们感谢赫尔辛基大学的Tero Ahola博士为建立这种MTase测定提供的帮助和建议。 我们感谢赫尔辛基大学在我们访问期间提供研究设施和支持。 这项工作得到中国国家自然科学基金资助(81130083,31170152和81271817)的支持。

参考文献

  1. Chen,Y.,Tao,J.,Sun,Y.,Wu,A.,Su,C.,Gao,G.,Cai,H.,Qiu,S.,Wu,Y.,Ahola,郭德华(2013)。 严重急性呼吸综合征冠状病毒RNA帽鸟嘌呤-N7-甲基转移酶的结构功能分析。 87(11):6296-6305。
  2. Chen,Y.,Su,C.,Ke,M.,Jin,X.,Xu,L.,Zhang,Z.,Wu,A.,Sun,Y.,Yang,Z.,Tien, Ahola,T.,Liang,Y.,Liu,X.和Guo,D。(2011)。 SARS冠状病毒RNA核糖的机制的生物化学和结构见解nsp16/nsp10蛋白复合物。 PLoS Pathog 7(10):e1002294。
  3. Chen,Y.,Cai,H.,Pan,J.,Xiang,N.,Tien,P.,Ahola,T.and Guo,D.(2009)。 功能屏幕显示SARS冠状病毒非结构蛋白nsp14作为新型帽N7甲基转移酶。 Proc Natl Acad Sci USA 106(9):3484-3489。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. and Guo, D. (2014). Biochemical Assays for MTase Activity. Bio-protocol 4(2): e1023. DOI: 10.21769/BioProtoc.1023.
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